Cross-linked hydrogels incorporating supramolecular host–guest structures serve as effective biosensor transducers utilizing their swelling capabilities to detect biomarkers. This study addresses the entire development chain of a novel biosensor. Initially, stable molecular compounds were synthesized and integrated into photo-cross-linkable polymers via spin coating. The resulting networks convert chemical recognition to detectable swelling signals triggered by competitive binding. When specific analytes are present, reversible supramolecular bonds break, releasing guest molecules that alter the gel polarity and induce swelling changes. To validate this mechanism, supramolecular interactions were rigorously analyzed. The polymers were then integrated into piezoresistive sensor setups. By comparing measuring and reference sensors, the system’s accuracy and reliability were demonstrated through the detection of spermine concentrations. These hydrogel-based sensors offer significant advantages: miniaturization for rapid response times, integrability, enhanced selectivity and sensitivity, and electrical outputs facilitating simple on-site diagnostics. The final sensor exhibited linear behavior within the physiologically relevant range (1–10 μM spermine) and achieved a detection limit of 0.39 μM. Consequently, an accurate, precise, and rapid on-site sensor was successfully created. Designed for high sensitivity, fast response, and long-term stability, these prototypes for biomedical spermine sensing hold promise for diverse applications, including noninvasive medical diagnostics, pharmaceuticals, healthcare, and environmental protection.